T H E J O U R N A L O F C E L L B I O L O G Y

Transcription

1 T H E J O U R N A L O F C E L L B I O L O G Y Supplemental material Craft et al., Figure S1. GFP -tubulin interacts with endogenous -tubulin. Ciliary matrix obtained from strain GFP-Tub1 and a wild-type control was incubated with anti- GFP (i.e., GFP nanobody or GFP binding protein) beads to purify GFP -tubulin and associated proteins. (A) Silver-stained gel showing the ciliary matrix (input), the unbound fraction (flow through), and the eluted fraction (eluate). GFP -tubulin, -tubulin, and the uncleaved ble-gfp -tubulin fusion protein are marked by arrowheads; the mean band intensity of the latter was 22% compared with that of cleaved GFP-tubulin (±8%, n = 7 flagellar preparations). (B) Western blot analysis of the fractions with the antibodies as indicated. Under the chosen stringent conditions (wash with 250 mm NaCl in HMEK), endogenous -tubulin did not coimmunoprecipitate with GFP-tubulin. (C) Comparison of soluble and insoluble ciliary tubulin. Western blot of axonemal and MM fractions isolated from strain GFP-Tub1 analyzed with the antibodies indicated. Loading was adjusted to present similar amounts of tubulin in both fractions. S1

2 Figure S2. Comparison of active transport and diffusion of fluorescent protein-tagged -tubulin. Gallery of kymograms showing active transport (open arrows in A D) and diffusion (open arrowheads in A E) of GFP -tubulin; IFT20-mCherry is shown in G and H (red ; the corresponding GFP-tubulin track is shown in F). Solid arrows in C and D: transition from IFT to diffusion indicative for unloading of GFP -tubulin; solid arrow in F: putative docking of GFP tubulin to the ciliary tip. White arrowheads in A, C, D, and I: reduced mobility of diffusing GFP -tubulin at the ciliary tip. Open arrowheads in I indicate bleaching events of mneongreen -tubulin. Bars, 1 µm and 1 s. S2

3 Figure S3. Western blot analysis of strains used in this study. (A) The C-terminal domain of GFP -tubulin is not required for incorporation into the axoneme. Western blot analysis of axonemes isolated from a wild-type control strain and strains expressing GFP -tubulin and the modified minuse, betac, and deltac GFP-tubulins. Modified tubulins were expressed in an ift20-1 IFT20-mCherry background. See Table S1 for a description of the constructs. (B) Western blot analysis of the fla10-1 GFP -tubulin and the ift20-1 IFT20-mCherry GFP -tubulin strains. Isolated cilia (fla) were fractionated and probed with the antibodies as indicated. (C) Western blot (top) and Coomassie stained gel (bottom) of whole cell extracts from wild type, shf2, lf2-1, and GFP -tubulin expressing version of these strains. Western blots were stained with anti-gfp and anti -tubulin. S3

4 Figure S4. Increased concentration of soluble tubulin in growing cilia. (A) Frequency of IFT20-mCherry particles in regenerating and steady-state cilia. The reduced apparent IFT frequency in this strain could be caused, for example, by low expression levels of IFT20-mCherry. Also, some IFT20-mCherry particles could have escaped detection due to their reduced brightness and/or loss of fluorescence. Error bars indicate SEM. (B) Western blot showing a dilution series of the axonemal fraction and the undiluted MM sample from regenerating cilia to determine the ratios of tagged and endogenous tubulin between the two fractions. The blot was probed with anti -tubulin, anti-gfp, and, as a control, an antibody to FAP12, the major membrane-associated protein in C. reinhardtii cilia. Compare to Fig. 1 D showing a similar blot of steady-state cells. (C) Quantitative analysis of the amount of tubulin in the MM fraction of steady-state (ss), regenerating (reg), and fully regenerated (fr) cilia. Quantifications are based on the blot shown in Fig. 6 A and band intensities were normalized for IC2 loading. This experiment was completed once. Elevated tubulin levels in the matrix of the fully regenerated cilia of the control might indicate that some cells in this sample have not yet completed regeneration. (D) Analysis of the GFP-tubulin transport frequencies in nonregenerating GFP-Tub1 derived L cilia and regenerating wild-type derived S cilia of long-short zygotes. Error bars indicate SEM. S4

5 Figure S5. Repeated FRAP of growing and nongrowing cilia is followed by similar rates of recovery. (A and B) Segments of a steady-state (A) and a regenerating (B) cilium were repeatedly bleached (indicated by arrowheads). Kymograms (top) and FRAP quantification (bottom) indicate similar rates (in percentage of pre-bleach GFP -tubulin fluorescence) of recovery after each bleaching step. (C and D) Individual frames (C) and kymograms (D) of a longshort cell. Bleached areas are marked by dashed circles. The kymogram (D) is a composite of several recordings, and arrowheads labeled a e indicate the positions of the frames in C. The time (in seconds) for each recording and the position the bleaching laser (Brackets) is indicated; overexposed frames caused by photobleaching were deleted. Arrows in D: GFP -tubulin trajectories. Note fast and strong recovery in subsequent bleachings of the short cilium while the initially bleached area in the long cilium remains visible. The extended observation time will also bleach some of the (axonemal) GFP -tubulin outside of the spot bleaching area. This loss of fluorescence in the nonbleached areas results in a higher apparent recovery in the bleached areas. Bars: (C) 2 µm; (D) 2 µm and 5 s. S5

6 Video 1. Anterograde transport of GFP -tubulin in C. reinhardtii cilia. TIRF imaging of a cilium of a cell stably expressing GFP -tubulin; the ciliary base is oriented to the bottom and the ciliary tip to top. The video corresponds to the kymogram in Fig. 2 A (a), was recorded at 10 fps, and is displayed in real time. Video 2. Diffusion of mneongreen -tubulin inside bleached cilia. After photobleaching of the cilia, images were recorded at high laser intensity to avoid accumulation of mneongreen -tubulin entering cilia by diffusion. Images were recorded using a microscope (Eclipse Ti-U) equipped with TIRF illumination at 31 fps. The video is displayed at real time and corresponds to Fig. 2 A (c) and Fig. S2 I. Video 3. GFP -tubulin is transported by IFT. Simultaneous recording of mneongreen -tubulin (left) and IFT20-mCherry (right) in partially bleached cilia. The video corresponds to Fig. 2 B, was recorded at 10 fps, and is displayed at 2. Images were recorded using a microscope (Eclipse Ti-U) equipped with TIRF illumination. S6

7 Video 4. Reduced mobility of diffusing GFP -tubulin near the ciliary tip. A GFP-tubulin particle moves by diffusion to the ciliary tip (top) and lingers near the tip with reduced mobility before reentering the ciliary shaft by fast pure diffusion. The video was recorded at 31 fps, is displayed at real time, and corresponds to Fig. 2 A (d). Images were recorded using a microscope (Eclipse Ti-U) equipped with TIRF illumination. Video 5. GFP -tubulin transport and incorporation during ciliary regeneration. This composite video is based on several recordings, spans 6 min 40 s, and shows a cell during regeneration of its two cilia after deciliation by a ph shock. The arrowheads mark the initial position of the ciliary tips. Note IFT trafficking of GFP-tubulin and its incorporation into the distal ciliary segment as cilia grow. The video corresponds to Fig. 3 B, was recorded at 10 fps, and is displayed as a 5 time lapse. Images were recorded using a microscope (Eclipse Ti-U) equipped with TIRF illumination. Video 6. GFP -tubulin transport by IFT in cilia of long-short cells. One of the two cilia was amputated by mechanical shear and the cell has initiated regrowth of the missing cilium. Then, both cilia of the long-short cells expressing GFP -tubulin and IFT20-mCherry were partially bleached. Note empty IFT-mCherry trains (red) in the long cilium (top), whereas GFP -tubulin and IFT20-mCherry largely co-migrate in the short cilium (bottom). The video relates to Fig. 4 B, was recorded at 10 fps, and is displayed as a 2 time lapse. Images were recorded using a microscope (Eclipse Ti-U) equipped with TIRF illumination. S7

8 Video 7. GFP -tubulin transport in cilia of a long-short zygote. A wild-type cell with regenerating cilia was fused to GFP- Tub1 cell (expressing GFP -tubulin) with full-length cilia, and all four cilia were partially photobleached. Intense IFT trafficking is limited to the regenerating cilia and incorporation of GFP -tubulin into the distal portions of those cilia is apparent. The video corresponds to Fig. 5 B, was recorded at 10 fps, and is replayed in real time. Images were recorded using a microscope (Eclipse Ti-U) equipped with TIRF illumination. Video 8. FRAP of GFP -tubulin in a steady-state cilium. A segment of the cilium was bleached using a focused laser beam. Entry of GFP -tubulin in the bleach area by diffusion results in limited recovery of fluorescence. The video was recorded at 10 fps and is displayed at 2.5. Images were recorded using a microscope (Eclipse Ti-U) equipped with TIRF illumination. Video 9. GFP -tubulin concentration in cilia of long-short cells. The distal segments of the short cilium (bottom) and the long cilium (top) are sequentially bleached using a focused laser beam. Note rapid FRAP, IFT, and incorporation of GFP -tubulin transport into the short cilium, whereas FRAP is weaker in the long cilium. The video corresponds to Fig. 6 E, is a composite of two 30-s recordings at 10 fps, and is displayed as a 6 time lapse. Images were recorded using a microscope (Eclipse Ti-U) equipped with TIRF illumination. S8

9 Table S1. The E-hook of -tubulin is not required for transport via IFT Construct C-ter ( ) IFT transport (µm/s) Incorporation into the axoneme sfgfp-tub1 LEKDFEEVGAAESAEGAGEGEGEEY 2.09 (±0.35, n = 114) Yes FP-minusE LEKDFAAVGAQSAQGAGAGAGAEY 1.83 (±0.36, n = 32) Yes FP-deltaC LEKDFEEVGAEY 2.04 (±0.43, n = 89) Yes FP-betaC LEKDFQQYQDASAEEEGEFEGEEEEAEF 2.29 (±0.57, n = 19) Yes The table shows the C-terminal sequences of wild-type and the modified -tubulin molecules. Transport velocities by anterograde IFT and qualitative assessment of axonemal incorporation of the strains are listed. The following Matlab source codes are also available as a zip file: tracksxml.m will read in data from an xml file generated by the Mosaic Particle Tracker; traj_anal.m to perform a quick analysis of particle track data, it will generate some values related to whether the particle is likely diffusing or moving at a constant velocity; traj_stats.m to perform diffusion analysis (mean square distance vs. time); and trajectories_ folder.m to perform diffusion analysis of a list of xml files generated by the Mosaic Particle Tracker. S9